Cryptosporidiosis, due to intestinal infection with the intracellular parasites Cryptosporidium parvum or C. hominis, is the leading cause of diarrhea resulting from waterborne outbreaks in the United States, and it is a major cause of pediatric infectious diarrhea in the developing world. The infection is chronic and often fatal for immunocompromised patients, and cryptosporidiosis causes as much as 50% of chronic diarrhea in AIDS patients. Unfortunately, nitazoxanide, the only drug with known efficacy, is only moderately effective for treatment of immunocompetent people, and it is equivalent to placebo in severely immunocompromised people. Cryptosporidiosis predominantly affects people in the developing world, and the high cost of de novo drug development is a major barrier to developing an effective treatment. Using a drug repurposing approach to address this economic barrier, the ultimate goal of this project is to develop an effective therapy for cryptosporidiosis The target product profile requires that the treatment be safe, low in cost, selectively toxic for Cryptosporidium at concentrations relevant within the small intestine and biliary tract, and capable of eliminating the parasite from patients in the absence of effective host immunity. Towards this end, preliminary data are presented documenting: 1) development and validation of a robust, cell-based, high-throughput screening (HTS) assay; 2) identification of FDA-approved drugs with selective anti-Cryptosporidium activity; and 3) an in vitro assay to compare the ability of different treatments to eliminate C. parvum from a cell-based tissue culture system. In aim 1, the abilities of the identified anti-Cryptosporidium drug leads to eliminate C. parvum will be determined in vitro, and then the drug leads will be tested in vivo using a model of cryptosporidiosis in severely immunocompromised mice. Synergistic drug combinations sometimes possess cidal antimicrobial activity (i.e., kill microbes rather than merely inhibit microbial growth) at achievable tissue concentrations despite static activity of each drug used alone, and, in some cases, drug combinations enable successful treatment of immunocompromised patients where either drug alone is ineffective. Therefore, in aim 2, combinatorial drug screens will be undertaken to identify drugs with synergistic anti-Cryptosporidium activity when used with nitazoxanide or the primary screening hits. As proof of concept, a screen was conducted in the presence of a sub-inhibitory concentration of nitazoxanide, which yielded five approved drugs with no anti-cryptosporidial activity when used alone that were strong C. parvum growth inhibitors in combination with low dose nitazoxanide. Synergy of promising drug combinations will be confirmed with checkerboard assays, and the most promising will be followed up in vitro and in vivo as in aim 1. Successful completion of these specific aims will result in identification of at least one FDA-approved drug or a combination of drugs that cures immunocompromised mice infected with C. parvum and is suitable for testing in human clinical trials.